Chemistry Reference
In-Depth Information
As expected, and as already shown in numerous studies [
3
,
10
,
11
,
16
,
17
,
23
,
24
,
26
-
32
,
34
-
41
], mixing the liposomes at normal ionic strength (20mM K-Hepes pH
7.4, 150mM KCl) resulted in robust lipid mixing. This is shown in Fig.
3.7
by the
plotting the fluorescence intensity of the donor fluorophore of the FRET pair as a
function of time. Clearly, if fusion proceeds, this signal decreases with time. As the
ionic strength was lowered progressively, substantial decrease in the lipid mixing
kinetics is seen (left panel of Fig.
3.7
) and membrane fusion is almost completely
blocked at low ionic strength (20mMK-Hepes pH 7.4, 5mMKCl, 300mM sucrose).
In low ionic strength buffers, osmolarity differences lead to disruption of liposomes
and therefore the osmolarity is compensated by adding sucrose to the buffer, which
increases the viscosity of the solution. However, the reduction in diffusion at higher
viscosities does not account for the complete blocking of membrane fusion. Indeed,
at an ionic strength of 5mMKCl, the debye screening length is about 25Å compared
to
7Å at normal ionic strengths.
Since the liposomes contained a high fraction of anionic lipids, the electrostatic
repulsion might be too high for the lipid membranes to come close enough for
SNARE-mediated membrane fusion to occur at low ionic strength. The experiments
were repeated with liposomes composed of pure zwitterionic lipids and also with
liposomes composed of a reduced (9:1) ratio of uncharged:charged lipids. As seen
in the right panel of Fig.
3.7
, membrane fusion does occur at lower ionic strengths.
∼
Fig. 3.7
SNAREmediatedmembrane fusion is blocked at low ionic buffer strengths due to repulsion
between charged lipids comprising the liposomes.
Left panel
Liposomes contain charged lipids PS
and Pi(4,5)P
2
. With the progressive reduction of the salt (KCl) concentration in the buffer solution,
the fusion is reduced, until it is almost completely blocked at a concentration of 5mMKCl (low ionic
strength). The experimental conditions are shown on the
top
.
Right panel
When only uncharged
lipids are used, the fusion proceed normally even when charges are not screened at low ionic
strengths of the buffer solution. The reduced kinetics at 5mM KCl concentration is due to the
increased viscosity of the buffer as described in the main text
